Reconstitutable microsphere compositions useful as ultrasonic contrast agents

ABSTRACT

Methods and suspensions are provided that are useful for preparing readily reconstitutable, dry compositions of micro- or nanospheres. The dry compositions find use in diagnostic applications such as ultrasonic imaging. The suspension includes as key ingredients one or both of t-butyl alcohol and/or an amorphous sugar (or mixture of amorphous sugars) in specified amounts that reduce aggregation of the particles comprising the suspension.

RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 60/517,219 filed Oct. 31, 2003,the disclosure of which is incorporated herein by reference in itsentirety.

BACKGROUND

Solid and hollow-cored micro- and nano-particles are used in a growingvariety of medical, pharmaceutical, and diagnostic applications. Wheninjected into the bloodstream, such particles can be used as ultrasonicechographic imaging contrast agents to aid the visualization of internalstructures, such as the heart and blood vessels. Such contrast agentsmay also be used to examine organ perfusion, for example, to assess thedamage caused by an infarct, to examine organs such as the liver, or todifferentiate between normal and abnormal tissues such as tumors andcysts.

Ultrasonic contrast is achieved when acoustic impedance between twomaterials at an interface is different. Ultrasonic imaging methods andparticle compositions useful as contrast agents are described in greaterdetail in, for example, Ultrasound Contrast Agents, Basic Principles andClinical Applications, Golderg et al., Eds, 2d Edition, 2001, MartinDunitz Ltd., Solid-cored particles (also called “matrix” particles)useful as ultrasound contrast agents are described in, for example, U.S.Pat. No. 5,558,857, U.S. Pat. No. 5,670,135, U.S. Pat. No. 5,674,468,U.S. Pat. No. 6,264,959, U.S. Pat. No. 6,177,062 and U.S. Pat. No.5,565,215. Hollow-cored particles useful as contrast agents aredescribed in, for example, U.S. Pat. No. 6,193,951, U.S. Pat. No.6,200,548, U.S. Pat. No. 6,123,922, U.S. Pat. No. 6,333,021, U.S. Pat.No. 6,063,362, U.S. Pat. No. 6,022,252, U.S. Pat. No. 6,569,405, U.S.Pat. No. 6,045,777 and currently pending U.S. application Ser. No.09/637,516. Both hollow- and solid-cored particles may also be used todeliver pharmaceutical products such as drugs and/or other therapeuticor diagnostic compositions to targeted organs or tissues in the body.Pharmaceuticals may be released from the particle by diffusion, bydegradation of the particle, or by rupture of the particle in situ usingultrasonic energy.

A well-known stabilization method for injectable ultrasonic contrastagents as well as for pharmaceutical delivery particles isfreeze-drying, also known as lyophilization. Various methods offreeze-drying and then stabilizing and storing a particle suspensionhave been previously described, for instance in U.S. Pat. No. 6,165,442.However, the particles in the suspension oftentimes aggregate during thelyophilization process (or upon storage). Such aggregation can beundesirable, especially in instances where the lyophilized particlecomposition will be administered to a patient via intravenous injection.

Aggregation problems are especially acute for particles composed ofproteins, or particles having a proteinaceous outer coating. Aggregationproblems can also be encountered with particles composed of syntheticpolymers and/or mixtures of synthetic polymers and proteins. Otherproblems inherent in the preparation and lyophilization of injectableparticles include removal of one or more of the organic solvents used inprocessing. This is particularly important in the formation andpreparation of hollow-cored particle compositions.

Therefore, there is a need for methods for improved preparation andhandling of compositions of lyophilized micro- and nano-particles toreduce aggregation and provide for more effectively and convenientlyreconstituted compositions.

SUMMARY

These and other needs are addressed by the present invention, which incertain aspects provides particle suspensions and methods for making dryparticle compositions that reduce the propensity of the particles toaggregate or “stick together” during lyophilization, storage, andreconstitution. Also provided are dry particle compositions that arereadily dispersible upon reconstitution with water.

The invention is based, in part, on two important discoveries. First,the Applicants have discovered that adding a specified quantity of anamorphous sugar to a suspension of polymeric particles comprising aproteinaceous outer coating greatly reduces the propensity of theparticles to stick together, especially during lyophilization of theparticle suspension. However, it was observed that if the amorphoussugar is present in concentrations sufficient to reduce or avoidaggregation of the particles upon lyophilization or storage of thelyophilized particle composition, removal of the solvents used in thefabrication of the particles is impeded. Second, the Applicants havediscovered that adding a specified quantity of t-butyl alcohol to aparticle suspension comprising an amorphous sugar in what wouldotherwise have been a sub-optimal concentration (low enough inconcentration to allow good solvent removal but too low to completelyinhibit aggregation) aids removal of solvents during lyophilization ofthe particle suspensions, and in particular aids the removal of solventsfrom the hollow core of hollow-cored particles and provides a stable,dry lyophilized particle composition with little or no aggregation ofthe particles. Dry particle compositions prepared from suspensionsincluding the specified quantities of t-butyl alcohol and/or amorphoussugar are readily dispersed upon reconstitution with water, making themideally suited for diagnostic and/or therapeutic applications. Becauseof this facile-dispersibility, dry particle compositions prepared bylyophilizing the particle suspensions described herein are especiallysuited for administration to animals and humans via intravenousinjection.

Thus, in one aspect, the present invention provides aqueous suspensionsof particles that are useful for preparing dry particle compositionssuitable for reconstitution and in vivo administration to animals andhumans that overcome the propensity of the particles to aggregate duringlyophilization as compared to conventional suspensions. The suspensiongenerally comprises from 0.3 to 4 mg of hollow-cored particles (weightis based upon the weight of the shell material) per milliliter (mL) ofsuspension or from 0.3 mg to 56 mg solid-cored particles per milliliterof suspension and one or both of the following: t-butyl alcohol and/oran amorphous sugar (or a mixture of two or more amorphous sugars). Theamounts of t-butyl alcohol and/or amorphous sugar(s) comprising thesuspension will depend upon whether the suspension compriseshollow-cored particles or solid cored particles. For sold-coredparticles, the suspension generally comprises t-butyl alcohol in aweight to weight ratio (t-butyl alcohol: particle) range ofapproximately 2.14:1 to 43:1 and/or an amorphous sugar (or mixture ofamorphous sugars) in a weight to weight ratio (total amorphous sugar(s):particle) range of approximately 0.02:1 to 0.86:1. For hollow-coredparticles, the suspension generally comprises t-butyl alcohol in aweight to weight ratio (t-butyl alcohol: particle) range ofapproximately 30:1 to 600:1 and/or an amorphous sugar (or mixture ofamorphous sugars) in a weight to weight ratio (total amorphous sugar(s):particle) range of approximately 0.3:1 to 12:1.

In general, the bulk of the suspension is water. However, the suspensionmay include additional solvents, such as the solvents and/or solventmixtures typically used during the preparation of the particles, and/orone or more excipients, such as, for example, buffering agents, agentsto adjust osmolality and tonicity and bulling agents. The suspensionsmay also include one or more surfactants. However, a significantadvantage of the suspensions described herein is the ability to handleand lyophilize the suspensions without significant aggregation of theparticles. Thus, while the suspensions may include surfactants and otherconventional anti-aggregation agents, the use of such agents is notnecessary.

In one embodiment, the suspension includes both the t-butyl alcohol andthe amorphous sugar(s).

In another aspect, the invention provides methods of making drycompositions of particles that are easily reconstitutable anddispersible upon addition of water. In one sense, the method compriseslyophilizing to dryness an aqueous particle suspension comprisingt-butyl alcohol and/or one or more amorphous sugars, as described above.The t-butyl alcohol and/or amorphous sugar(s) (and any optionalexcipients and/or surfactants) are typically added to a particlesuspension after the formation of the polymeric and/or proteinaceousparticles and prior to lyophilization. For example, solid-cored orhollow cored particles can be prepared using conventional techniques,combined with an aqueous excipient composition including the t-butylalcohol, amorphous sugar(s) and/or any desired optional excipientsand/or surfactants in concentrations suitable to yield an aqueoussuspension of particles as described above, and this suspensionlyophilized to dryness. If desired, the particles can be collected byfiltration or other means (e.g., centrifugation) prior to mixing withthe aqueous excipient composition. If desired or necessary, solventexchange prior to mixing with the aqueous excipient composition can beaccomplished without collecting the particles by, for example,diafiltration or other conventional means.

Although the method can be used with virtually any type of particlesthat have a propensity to aggregate and/or stick together, it has beendiscovered that the method is especially advantageous in the preparationof dry compositions of bilayered, hollow-cored particles, such as thebilayered protein coated polymeric nano- and/or micro-particlesdescribed in U.S. Pat. No. 6,193,951 and co-pending U.S. applicationSer. No. 09/637,516 (WO 01/12071), the disclosures of which areincorporated herein by reference.

In a specific embodiment of the method, both t-butyl alcohol and one ormore amorphous sugars are added to an aqueous suspension of such formed,bilayered, hollow-cored particles, either alone or in combination withone or more excipients, prior to lyophilization of the suspension. Thesuspension is then lyophilized to dryness to yield a dried particlecomposition that is readily dispersible upon addition of water. As isknown in the art, the hollow-cored particles comprising the drycomposition may be filled with a specified gas or mixtures of gases,such as nitrogen (N₂), air, or a perfluorocarbon, by filling thelyophilization chamber containing the dry particle composition with thespecified gas or gases.

In another aspect, the present invention provides dry, readilydispersible and/or reconstitutable compositions of particles. The drycompositions are formed by lyophilizing an aqueous suspension ofparticles comprising t-butyl alcohol and/or an amorphous sugar(s) asdescribed herein, and generally comprise particles and an amorphoussugar or mixture of two or more amorphous sugars in a weight ratio rangeof about 0.3:1 to 12:1 (for hollow-cored particles) or 0.02:1 to 0.86:1(for solid-cored particles). The dry composition may optionally includeone or more excipients and/or surfactants, as described above. Theexcipients may be included in the suspension prior to lyophilization, orthey may be added to the dry, lyophilized composition. When included inthe composition, such excipients are typically used in amounts commonlyemployed in particle compositions designed for therapeutic and/ordiagnostic applications. In a specific embodiment, the compositioncomprises the following components with the indicated approximate weightto weight ratios (wt ingredient: wt particle):

wt Ratio Particles Ingredient hollow cored solid cored Sucrose, NF 1.5:10.11:1 Polyethylene Glycol 3350, NF 17.3:1  1.24:1 Poloxamer 188, NF3.6:1 0.26:1 Glycine, USP 7.2:1 0.52:1

The dry composition may be packaged in any convenient packagingcontainer, depending upon the particular application. For example, thedry composition may be packaged in bulk, permitting desired quantitiesto be measured out on an as-needed basis. Typically, the dry compositionwill be packaged in single use quantities in sealed glass vials of asize and configuration suitable for reconstituting the composition withwater directly in the vial so that sterile conditions can be maintained.Vials of hollow-cored, gas-filled particles may be stored in the vialsor other similar containers under a headspace containing the fillergas(es) such that the gas(es) in the cores do not diffuse out duringstorage.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A provides a bar graph illustrating the peak diameters ofhollow-cored microsphere compositions prepared as described in Example1;

FIG. 1B provides a bar graph illustrating the mean diameters ofhollow-cored microsphere compositions prepared as described in Example1;

FIG. 1C provides a bar graph illustrating the median diameters ofhollow-cored microsphere composition prepared as described in Example 1;

FIG. 1D provides a bar graph illustrating the volume percentage ofmicrospheres having diameters greater than 7 microns for hollow-coredcompositions prepared as described in Example 1;

FIG. 2A provides a bar graph illustrating the peak diameters ofsolid-cored microsphere compositions prepared as describe in Example 2;

FIG. 2B provides a bar graph illustrating the mean diameters ofsolid-cored microsphere compositions prepared as described in Example 2;

FIG. 2C provides a bar graph illustrating the median diameters ofsolid-cored microsphere compositions prepared as described in Example 2;and

FIG. 2D provides a bar graph illustrating the volume percentage ofmicrospheres having diameters greater than 10 microns for solid coredcompositions prepared as described in Example 2.

DETAILED DESCRIPTION

The present invention provides methods and suspensions for formingcompositions of particles that are less susceptible to particleaggregation than currently available methods and/or compositions. Themethods and suspensions are useful in forming particle compositions foruse in diagnostic imaging, drug delivery, and other medical andpharmaceutical applications. Also provided are dried particlecompositions formed by the methods. Such dry compositions are readilydispersible in water, making them ideally suited for diagnostic andtherapeutic applications. The methods are particularly advantageous forhandling suspensions of particles comprising polymers and/or proteins,as well as other particles that have a propensity to aggregate or “sticktogether” during lyophilization, storage, and/or reconstitution. Amongtheir numerous potential applications, the methods and suspensions areuseful in the preparation of solid-cored or “matrix” particlescomprising polymers and/or proteins, such as those disclosed in, forexample, U.S. Pat. No. 5,558,857, U.S. Pat. No. 5,670,135, U.S. Pat. No.5,674,468, U.S. Pat. No. 6,264,959, U.S. Pat. No. 6,177,062 and U.S.Pat. No. 5,565,215, and hollow-cored particles comprising polymersand/or proteins, such as those disclosed in U.S. Pat. No. 6,193,951,U.S. Pat. No. 6,200,548, U.S. Pat. No. 6,123,922, U.S. Pat. No.6,333,021, U.S. Pat. No. 6,063,362, U.S. Pat. No. 6,022,252, U.S. Pat.No. 6,569,405, U.S. Pat. No. 6,045,777 and in co-pending U.S.application Ser. No. 09/637,516 (WO 01/12071). The particles, whethersolid-cored or hollow-cored, will typically have diameters in a sizerange suitable for passing through the circulatory system (and avoidingaccumulation by the RES). Typically, the diameters of the particles willbe less than 10 μm, and the collection of the particles comprising acomposition will have a relatively narrow distribution of averagediameters. Particles suitable for administration via intravenousinjection typically will have a numeric mean diameter in the range of3-3.5 microns, with greater than 95%, and preferably greater than 99%,having diameters of less than 7 microns.

The methods are broadly applicable to the preparation of drycompositions containing a wide variety reconstitutable particles. Ingeneral a method is provided for preparing aqueous particle suspensionsin which particle aggregation problems are substantially reduced. Thismethod is useful for any particles that have a propensity to aggregateduring lyophilization, storage, and/or reconstitution, and generallyinvolves lyophilizing to dryness an aqueous suspension comprising fromabout 0.3 mg to 4 mg of hollow-cored particles (weight is based upon theweight of the shell material) per milliliter of suspension or from about0.3 mg to 56 mg solid-cored particles per milliliter of suspension andone or both of t-butyl alcohol and an amorphous sugar (or mixture of twoor more amorphous sugars) in specified weight to weight ratios,depending upon whether the suspension comprises hollow-cored orsolid-cored particles. For hollow-cored particles, the amorphous sugars)is typically included in the suspension at a weight to weight ratio(total weight amorphous sugars: wt particles) in the range of about0.3:1 to 12:1 and/or t-butyl alcohol is included in the suspension at aweight to weight ratio (wt t-butyl alcohol:wt particles in the range ofabout 30:1 to 600:1. In a specific embodiment, the weight to weightratio of total amorphous sugar(s) is in the range of about 0.75:1 to 3:1and/or the weight to weight ratio of t-butyl alcohol is in the range ofabout 60:1 to 150:1.

For solid-cored particles, the amorphous sugar(s) is typically includedin the suspension at a weight to weight ratio in the range of about0.02:1 to 0.86:1 and/or t-butyl alcohol is included in the suspension ata weight to weight ratio in the range of about 2.14:1 to 43:1. In aspecific embodiment, the weight to weight ratio of total amorphoussugar(s) is in the range of about 0.07:1 to 0.36:1 and/or the weight toweight ratio of t-butyl alcohol is in the range of about 4:1 to 11:1.

In a specific embodiment, the suspension includes both t-butyl alcoholand an amorphous sugar(s) in the disclosed weight to weight ratio.

“Amorphous sugars,” as used herein, are those sugars that, while capableof crystallizing, can be trapped in a non-crystalline, amorphous statewhen lyophilized. These lyophilized amorphous sugars can spontaneouslyconvert to a crystalline form if exposed to temperatures in excess oftheir glass transition temperature (T_(g)). Therefore, amorphous sugarsuseful in the present invention are those that have a relatively highglass transition temperature (T_(g)), typically above approximately 20°C. Specific examples of amorphous sugars suitable for use as describedherein include, but are not limited to, sucrose, trehalose and lactose.The amorphous sugar(s) and/or t-butyl alcohol may be added during theparticle formation or purification process as an additive in one or moreof the solutions carrying the particles or other aqueous compounds thatform the particles. More typical is for the amorphous sugar(s) and/ort-butyl alcohol to be added as part of an excipient or compositionsolution that is combined with a particle suspension after particleformation.

The amorphous sugar(s) in the disclosed concentration ranges functionsas an aggregation inhibitor in the lyophilization, storage, and/orreconstitution processes. At an amorphous sugar concentrationsubstantially below the disclosed range, particulate aggregation maycause problems in forming a composition of discrete, reconstitutableparticles. If the amorphous sugar concentration is too high, solventremoval may be unacceptably hindered. Solvent removal difficulties are asubstantial concern in the preparation of hollow-cored particles, suchas for instance bilayered, hollow-cored protein-coated polymericparticles, as described in U.S. Pat. No. 6,193,951 and co-pending U.S.application Ser. No. 09/637,516 (WO 01/12071).

The use of t-butyl alcohol in the disclosed concentration rangesprovides dual benefits. In the disclosed concentration ranges, t-butylalcohol acts to reduce the tendency of the particles to aggregate whileenhancing solvent removal. The reduced aggregation effect is mostpronounced when t-butyl alcohol is used in combination with an amorphoussugar as described above. In general, t-butyl alcohol has propertiesthat result in it being almost completely removed from the suspensionduring lyophilization, making it particularly advantageous for use as anon-aggregation agent in making dry particle compositions suitable forin vivo administration to animals and humans. DMSO, or a mixture of DMSOand t-butyl alcohol, may also be used to similar effect.

Additional excipients may also be added to the aqueous particlesuspension, either as further constituents of an excipient solutionadded after particle formation or during or before the particleformation step or steps. These excipients may include surfactants, suchas poloxamers or tweens; bulking agents such as mannitol, lactose, orglycine; buffering agents such as acetate, citrate, or phosphate;collapse temperature modifiers such as dextran, polyethylene glycol, orsugars; crystalline matrix components such as mannitol or glycine;tonicity and osmolality modifiers such as mannitol, glycine, or sodiumchloride, among others.

The formed particle suspension containing an amorphous sugar and/ort-butyl alcohol in the above-disclosed weight ratio ranges may belyophilized to from a dry, reconstitutable particle composition.Lyophilization removes a substantial fraction of the water and thet-butyl alcohol and other processing solvents that may be present eitherin the suspension or within the particles. Hollow-cored particles can befilled with a gas or mixture of gases by flooding the lyophilizationchamber with the gas(es). The lyophilized composition may beconveniently stored and/or transported in vials or some other suitablecontainer. If the particles are gas-filled, they can be stored under thegas(es) used to fill the particles. Prior to use, the composition may bereconstituted with water to form a discrete suspension of particleshaving a physiologically compatible osmolality and pH.

In one embodiment, the reconstituted suspension advantageously comprisessuspended particles in a concentration range of approximately 0.3 to 6mg of hollow-cored particles or 0.3 to 84 mg solid-cored particles permilliliter (mL) (for hollow-cored particles the weight is based uponparticle shell weight). In another embodiment, the aqueous particlesuspensions, dry lyophilized particle compositions and reconstitutedparticle compositions include, in addition to the particles, amorphoussugar(s) and/or t-butyl alcohol (for the aqueous particle suspension),glycine, polyethylene glycol, and/or poloxamer 188 in the followingconcentration ratios:

wt:wt Ratio (wt excipient:wt particles) Hollow-cored Solid-coredExcipient particles particles Glycine 0:1 to 75:1 0:1 to 6:1polyethylene glycol (MW 2000 to 6000) 6:1 to 300:1 0.4:1 to 22:1poloxamer 188 0:1 to 60:1 0:1 to 4.5:1

The methods described herein are of particular advantage in preparinghollow-cored, bilayer polymeric particles having a proteinaceous outerlayer or shell, such as those described in U.S. Pat. No. 6,193,951 andin co-pending U.S. application Ser. No. 09/637,516 (WO 01/12071), thedisclosures of which are incorporated herein by reference. The specific,exemplary applications described below are focused on injectablecompositions of microparticles and nanoparticles as described in thesereferences. In general, however, any type of particle suspension, and inparticular any biologically-compatible particle suspension, that issusceptible to problems caused by undesirable particle aggregation maybe prepared as described herein.

In one exemplary embodiment, the particles comprising the lyophilizableaqueous suspension and dry, reconstitutable composition have a bilayeredshell enclosing a hollow core. The outer layer of the shell may beformed of a protein or other biologically compatible amphiphilicmaterial, such as, for instance, cross-linked albumin. The outer layerforms the surface of the particle which is exposed to the blood andtissues win the body. The inner layer may be a synthetic polymer or asynthetic biodegradable polymer, such as, for instance,poly(D,L-lactide). For use as ultrasound contrast agents, the cores ofthe particles may be filled with a gas, such as air, nitrogen or aperfluorocarbon. Particles are constructed such that the majoritycomprising the suspension or composition will have diameters within therange of about one to ten microns in order to pass through the capillarysystem of the body. Alternatively, the particles may be constructed withdiameters below 1 μm, such as for instance in the range of 200 to 800nm, for use in imaging of, or delivering a pharmaceutically active agentto, the lymph node system.

Since these particles have a shell comprising an outer and inner layer,the layers may be tailored to serve different functions. The outer,exposed layer serves as the biological interface between the particlesand the body. The outer layer therefore generally comprises abiocompatible material that may be amphiphilic—having both hydrophobicand hydrophilic characteristics. The outer layer may also be formed ofone or more synthetic biodegradable polymers. In addition to beingamphiphilic, the outer layer may also have chemical features that permitcharge and chemical modification. The inner layer comprises abiodegradable polymer, which may be a synthetic biodegradable polymer.The inner layer provides or enhances mechanical or drug deliveryproperties to the particle which may not be sufficiently provided by theouter layer. Because the outer layer provides a biologically compatibleinterface, selection of the polymer may be made without beingconstrained by surface property requirements. The polymer may beselected for its modulus of elasticity and elongation, which define thedesired mechanical properties. Typical biodegradable polymers suitablefor use as the inner layer of such bilayered particles are described inU.S. Pat. No. 6,193,951 and co-pending U.S. application Ser. No.09/637,516 (WO 01/12071), the disclosures of which are incorporatedherein by reference. Additional suitable biodegradable polymers aredescribed in Langer, et al. (1983) Macromol Chem. Phys. C23, 61-125,incorporated herein by reference. These various polymers can also beused to make solid-cored particles, which can be optionally coated withan outer layer of biocompatible, optionally amphiphilic, material, asdescribed above.

For particles used as ultrasonic contrast agents or as a targeted,ultrasonically released drug carrier agent, the inner layer typicallyhas a thickness no greater than that necessary to meet the minimummechanical or drug carrying/delivering properties. This maximizes theinterior gas volume of the particles. The greater the gas volume withinthe particles the better their echogenic properties. The combinedthickness of the outer and inner layers of the particles depends, inpart, on the mechanical and drug carrying/delivering properties requiredof the particles, but typically the total shell thickness will be in therange of 10 to 750 nm.

Briefly, these particles may be formed by a method comprising thefollowing general steps. Two solutions are prepared, the first being anaqueous solution of the outer layer biomaterial. The second is asolution of the polymer (“polymer solution”) which is used to form theinner layer, in a relatively volatile water-immiscible liquid which is asolvent for the polymer (“polymer solvent”), and a relativelynon-volatile water-immiscible liquid which is a non-solvent for thepolymer (“polymer non-solvent”). The polymer solvent is typically aC5-C7 ester, such as isopropyl acetate. The polymer non-solvent istypically a C6-C20 hydrocarbon such as decane, tridecane, cyclohexane,cyclooctane, and the like. In the polymer solution, the polymer and thewater-immiscible solvents are combined so that the polymer fullydissolves and the two solvents are miscible with agitation. The polymersolution (organic phase) is slowly added to the biomaterial solution(aqueous phase) with agitation to form an emulsion. The relativeconcentrations of the solutions and the ratio of organic phase toaqueous phase utilized in this step and the degree of agitationessentially determine the final particle size and shell thickness. Afterthorough mixing of the emulsion, it is dispersed into water andtypically warmed to about 30-35° C. with mild agitation. A cross linkingagent, for example a carbodiimide or a bifunctional aldehyde such asglutaraldehyde, is added to the mixture to react with the biomaterialenvelope to render it water insoluble, stabilizing the outer layer.

The inner core of the newly formed outer layer contains a solutioncomprising the polymer, the polymer solvent and the polymer non-solvent,each of which have different volatilities. As the more volatile polymersolvent evaporates or is diluted, the polymer precipitates in thepresence of the less volatile polymer non-solvent. A film of precipitateis thus formed at the interface with the inner surface of thebiomaterial (outer) layer. This precipitate forms the inner layer of theparticle as the more volatile polymer solvent is reduced inconcentration either by dilution, evaporation, or the like. The core ofthe formed particle contains predominately the polymer non-solvent.

At this stage, the formed particles are collected and formulated into anaqueous suspension including one or both of t-butyl alcohol and one ormore amorphous sugars at the disclosed concentration ranges forhollow-cored particles, as well as any optional desired excipientsand/or surfactants. The aqueous suspension may be prepared by suspendingformed particles collected by centrifugation, filtration or other meansin an aqueous solution comprising the desired amounts of t-butylalcohol, amorphous sugar(s), and optional excipients and surfactants.Alternatively, the solvent system of the formed particles can be changedto a suspending medium by, for example, diafiltration or dilution (orother means or combination of means) and the t-butyl alcohol, amorphoussugar(s) and any desired excipients and/or surfactants dissolved in theaqueous solvent system to provide an aqueous particle suspensionaccording to the invention.

This aqueous suspension is then dried by lyophilization, yielding a dry,reconstitutable particle composition that is typically in the form of alyophilized cake. Inclusion of the amorphous sugar in the aqueousparticle suspension that gets lyophilized minimizes particle aggregationin the lyophilized product. Inclusion of t-butyl alcohol further detersparticle aggregation that occurs after reconstitution of thelyophilized, dry composition. The amorphous sugar remains in the dry,lyophilized particle composition, while the bulk of the t-butyl alcoholis removed. Use of these additives, whether during particle formation,processing of the suspension or as excipients added to a suspension ofparticles just prior to lyophilization, tends to provide a lyophilizedcake having a high porosity and surface area. These additives may alsoincrease the drying rate during lyophilization by providing channels forwater and solvent vapor to be removed. They may also provide alyophilized cake having a higher surface area than a lyophilized productprepared without them, which is beneficial in later reconstitutionsteps.

As previously disclosed in U.S. Pat. No. 6,193,951 and co-pending U.S.application Ser. No. 09/637,516 (WO 01/12071), aggregation of thesebilayered particles during formation may be further minimized bymaintaining a pH of at least one to two pH units above or below theisoelectric point (P_(i)) of the biomaterial forming the outer surface.As an alternative, the particles may be formulated at or near the P_(i)with the use of surfactants to stabilize against excessive aggregation.In any event, buffer systems of the dry, lyophilized composition to beinjected into the subject should be physiologically compatible.

The dry, lyophilized particle composition may be provided in unitcontainers containing a total weight in the range of approximately 1 to50 mg of hollow-cored particles or 1 to 700 mg of solid-cored particlesper container. Particles for use as ultrasonic contrast agents forimaging the circulatory system typically have a mean diameter ofapproximately 3 microns with the size range of approximately 1 and 10microns. Typically, less than 5% of the particles will have a diametergreater than approximately 10 microns. Alternatively, particles forultrasonically imaging the lymphatic system may have average diametersin the range of approximately 200 to 800 nm as described in co-pendingU.S. application Ser. No. 09/637,516 (WO 01/12071).

Particles in a specific example of the present invention have an outerlayer of cross-linked albumin. The albumin may be human serum albumincross-linked with a dialdehyde cross-linker, such as glutaraldehyde Theparticles also have an inner layer of poly(D,L-lactide) thatencapsulates a hollow core which may be filled with a gas or mixture ofgases (e.g., air, nitrogen, perfluorocarbons, etc.). For applicationssuch as delivery of a drug or some other pharmaceutically active agent,the core may be filled with the drug. Alternatively, the inner layer mayfarther comprise the drug if it is co-precipitated with thebiodegradable polymer during formation of the inner layer as describedbelow.

In a specific embodiment, the glutaraldehyde crosslinkedalbumin/polylactide particles are formulated into an aqueous suspensioncomprising t-butyl alcohol in the disclosed weight ratio, sucrose in thedisclosed weight ratio and polyethylene glycol, glycine and a poloxamer(at weight ratios discussed further below) such that afterlyophilization the particles in the dry, lyophilized composition arecontained within a matrix of polyethylene glycol, glycine, sucrose andthe poloxamer, such as for instance poloxamer 188. Poloxamer is anon-proprietary name used in conjunction with a numeric suffix forindividually unique identification of products for which a food, drag orcosmetic use is likely.

Upon reconstitution, the product would typically contain approximately0.3 to 6 mg of hollow-cored particles or 0.3 to 84 mg of solid-coredparticles per milliliter, however it is understood that it is possibleto add any amount of reconstitution media to provide a range ofconcentrations beyond what is disclosed herein. The reconstitution mediafurther may be isoosmotic such that the final osmolality of thereconstituted product is essentially independent of the volume ofreconstitution media used.

In a specific embodiment of the formation of the hollow-cored particleshaving an outer layer of crosslinked albumin and an inner layer of poly(D,L-lactide), a pH-adjusted aqueous solution containing the albumincomprising the outer layer is first prepared. In one embodiment, the pHis in the range of approximately 3 to 9, more specifically approximately4. The albumin may be human serum albumin. The pH may be adjusted byaddition of an acid, for example hydrochloric acid. The albuminconcentration is typically in the range of approximately 4% to 10% byweight. Monodisperse emulsions are favored at concentrations aboveapproximately 4% albumin by weight. Aggregation of the resultantparticles may become a problem at concentrations above about 10% albuminby weight.

Next, an organic solution containing poly(D,L-lactide) and cyclooctane(polymer non-solvent) dissolved in isopropyl acetate (polymer solvent)is prepared and emulsified into the aqueous solution. In specificembodiments, the intrinsic viscosity of the poly(D,L-lactide) should begreater than about 0.15 dL g⁻¹ (0.5% in chloroform, 30° C.) to maintainthe particle integrity. The concentration of the poly (D,L-lactide) isin the range of approximately 0.2 to 3% by weight of the solution tomaintain a sufficient particle wall strength without causing excessdifficulty in removing the cyclooctane polymer non-solvent duringlyophilization. The ratio of isopropyl acetate to cyclooctane is in therange of approximately 30:1 to 3:1 by weight. The higher ratios favorthicker and/or stronger particle walls. However, use of too high a ratiomay result in walls that are so thick that formation of the hollowparticle core is impaired. Use of excessive cyclooctane may result inoverly fragile particle walls that may rupture in the hydrostaticenvironment of the circulatory system.

The organic solution is emulsified into the aqueous solution usingstandard emulsification techniques, such as membrane emulsification.Typically, the emulsification is performed at about 30° C. under flowrate and pressure conditions sufficient to provide a droplet size ofabout 4 microns (volumetric). The organic to aqueous component ratio isin the range of approximately 0.3:1 to 3:1, and more typicallyapproximately 1.62:1. Ratios near the upper end of this range favorparticulate monodispersivity. However, the use of an excessivelyelevated ratio may result in an emulsion that is too thick forprocessing. Below the lower ratio, the volume of the container requiredmay become a limiting factor, although if suitable containers areavailable, lower ratios may be used.

The emulsion is then diluted approximately 3 to 18-fold, preferablyabout 4-6 fold (with stirring) into a second aqueous solution containinga cross-linker, such as glutaraldehyde. The crosslinker is included inthe second aqueous solution at a concentration sufficient to provide aweight to weight ratio (crosslinker: albumin) in the resultant dilutedsuspension in the range of about 0.05:1 to 1:1. For glutaraldehyde, afinal crosslinker to albumin weight ratio in the range of about 0.2:1yields good results. The pH of this aqueous solution may be adjusted toa desired usage, such as for example a pH in the range of about pH 6 to10, preferably in the range of about pH 7-8.

Following dilution, stirring is continued at 30° C. until the isopropylacetate is substantially removed by evaporation. Poloxamer 188 is thendissolved into the aqueous suspension, typically, to a concentration ofabout 0.25% by weight.

The suspension is then terminally filtered to remove aggregates andpolymeric debris and diafiltered with aqueous poloxamer 188 solution(0.25% by weight) to remove unreacted glutaraldehyde and unassociatedalbumin. The volume of the suspension may be adjusted by dilution withthe aqueous poloxamer 188 solution to achieve the desired particleconcentration range of 0.09 wt % to 1.2 wt % (0.9 to 12 mg/mlsuspension). In a specific embodiment, the particle concentration isadjusted to 0.5 wt %.

A concentrated aqueous excipient solution is prepared separately andadded to the particle suspension to yield an aqueous particle suspensionaccording to the invention. The aqueous excipient solution containst-butyl alcohol and/or one or more amorphous sugar(s), in a specificembodiment sucrose, at concentrations sufficient to provide resultantweight to weight ratios (wt ingredient: wt particles) of 30:1 to 600:1(t-butyl alcohol) and 0.3:1 to 12:1 (sucrose), as previously described.In a specific embodiment, the aqueous excipient solution includes botht-butyl alcohol and sucrose at weight to weight ratios of 105:1 and1.5:1, respectively.

The aqueous excipient solution may further include one or moreexcipients and/or surfactants, as discussed above. In a specificembodiment, the concentrated aqueous excipient solution additionallyincludes polyethylene glycol (PEG) having an average molecular weight inthe range of approximately 2200 to 8000 (preferably about 3400; PEG3350), a poloxamer preferably poloxamer 188) and glycine inconcentrations sufficient to yield weight to weight ratios (ingredient:particles) in the resultant aqueous suspension in the range of about 6:1to 300:1 (BEG), 0:1 to 60:1 (poloxamer) and 0:1 to 75:1 (glycine),respectively. In a specific embodiment, these excipients arc included inthe concentrated aqueous excipient solution to yield weight to weightratios in the resultant aqueous particle suspension of 17.3:1 (PEG),3.6:1 poloxamer) and 7.2:1 (glycine), respectively.

The particle suspension and concentrated excipient solution are combinedunder chilled conditions in a proportion of approximately 1 partsuspension to 2 parts concentrated excipient solution. The suspension isthen dispensed into containers such as vials, lyophilized to dryness andstoppered under reduced nitrogen pressure. The vials typically contain auseful unit amount of particles, typically from about 2 to 200 mg ofhollow-cored particles or 2 to 2800 mg solid-cored particles per gram ofdry, lyophilized composition.

The lyophilized composition may be reconstituted by addition of water(or other physiologically acceptable buffer) to form a physiologicallyacceptable, injectable suspension of microparticles having an osmolalityin the range of approximately 200 to 300 mOs/kg. The dry, lyophilizedcomposition according to this embodiment which includes hollow-coredparticles has the following concentration ratios of its components:

Ingredient wt./Particle wt. Ratio specific Ingredient Low highembodiment Polyethylene Glycol, NF 6:1 300:1  17.3:1  Poloxamer NF 0:160:1 3.6:1 Amorphous sugar 0.3:1   12:1 1.5:1 Glycine, USP 0:1 75:17.2:1

A typical dry, lyophilized composition including a useful unit amount ofhollow-cored particles may have the following composition:

Ingredient mg/vial % w/w Polylactide/Albumin Particles 5.0 3.3Polyethylene Glycol 3350, NF 86.7 56.6 Poloxamer 188, NF 18.0 11.7Sucrose, NF 7.5 4.9 Glycine, USP 36.0 23.5 Total 153.2 100.0

Vials or other closed and/or sealed vessels containing the dry,lyophilized particle composition have a good shelf life and are easilyreconstitutable with water to form an injectable ultrasound imagingagent. For hollow-cored particles, the reconstituted suspension maycontain the following ingredients in the following concentrations:

Ingredient mg/ml Polylactide/Albumin Particles 1.5-2.5 PolyethyleneGlycol 3350, NF 43.35 Poloxamer 188, NF 9.0 Sucrose, NF 3.75 Glycine,USP 18.0 Water for injection, USP qs

The reconstituted product is injected preferably by bolus or by infusioninto the blood steam of the subject and then used in conjunction withone or more methods for diagnostic imaging and/or targeted drug orpharmaceutical delivery.

EXAMPLES

The following examples are provided by way of illustration and are notintended to limit the invention.

Example 1

This example demonstrates the ability of the amorphous sugar sucroseand/or t-butyl alcohol to reduce aggregation of hollow-coredglutaraldehyde crosslinked albumin/polylactide microspheres duringlyophilization and reconstitution.

Preparation of cyclooctane-filled hollow-cored albumin/polylactidemicrospheres. An organic solution containing 48.4 gm poly (D,L-lactide)(inherent viscosity of 0.41 dL/gm at 0.5% in chloroform, 30° C.), 0.666kg cyclooctane, and 4.450 kg isopropyl acetate was prepared bydissolution of the polymer in the solvent mixture. The organic solutionwas slowly added with stirring to 3.25 kg of a 5 wt % solution of USPgrade human serum albumin which had been adjusted to a pH of 4.0 with10% HCl. While maintaining a temperature of 30° C., the resultingmixture was circulated through a sintered stainless steel fit. Thisprocess yielded an oil-in-water emulsion having an average volumetricdroplet size of about 4 microns. An aqueous solution containing 30 kg ofa 0.1% aqueous solution of glutaraldehyde was prepared. The pH wasadjusted to between 7.2 to 8.0 using 1N NaOH. Approximately 6.8 kg ofthe emulsion was next added with stirring to the bath. Stirring of thebath was continued at 30° C. with a stream of dry nitrogen gas passingover the mixture until the isopropyl acetate was substantially removedby evaporation (overnight). After removal of the isopropyl acetate, thesuspension was cooled to 18° C. and poloxamer 188 was added to theresultant suspension in the amount sufficient to yield a finalconcentration of 0.25 wt %. The suspension was depth-filtered to removemicrocapsule aggregates and polymeric debris. To remove excessglutaraldehyde, formed salts, and the unassociated albumin, thesuspension was next concentrated down and then washed by diafiltrationagainst approximately 7 volumes of a 0.25 wt % aqueous solution ofpoloxamer 188 using a 0.65 micron hollow fiber TFF. The diafilteredsuspension was diluted with aqueous poloxamer 188 (0.25 wt %) to yield asuspension having a microsphere concentration of 5 mg microsphere shellweight per gram of suspension. The size distribution of the microspheresin the diluted suspension was measured with a Malvern 2000 particle sizeanalyzer and found to have a volumetric peak diameter of 3.86 microns.

Hollow Microsphere Formulation and Lyophilization. Separately, fourdifferent aqueous solutions were prepared to serve as lyophilizationexcipients using ingredients and at concentrations (by weight) inaccordance with the table below.

Lyophilization Formulation Designation Excipient Solution 1 2 3 4tert-butyl alcohol 26.25%  26.25%    0%   0% Polyethylene glycol 4.34%4.34%  4.34% 4.34%  Glycine  1.8% 1.8%  1.8% 1.8% Poloxamer 188  0.9%0.9%  0.9% 0.9% Sucrose 0.38%   0% 0.38%   0% Deionized water 66.3%66.7%  92.6% 93.0% 

The diluted microsphere suspension was next formulated with the 4prepared excipients at a ratio of 1 part suspension to 2 parts excipientsolution by weight. The resulting formulations were each dispensed into10 ml serum vials at 3 ml/vial and then lyophilized to a dry cake usingan FTS Dura-Stop lyophilizer and capped under nitrogen. During thislyophilization process, the cyclooctane core of the microspheres wasremoved to render hollow nitrogen-filled microspheres.

Vials of the now dried suspension were reconstituted in 2 ml deionizedwater and the size distribution of the microspheres in the suspensionswere next determined using a Malvern 2000 particle size analyzer.Results of the size measurements are shown in the table below. Thederived statistics in the table are based upon a volumetric frequencyhistogram of microsphere size and represent an average over three vials.

Formu- Formu- Formu- Formu- lation #1 lation #2 lation #3 lation #4 ModeDiameter 3.68 μm 3.86 μm 4.07 μm 5.21 μm Mean Diameter 3.78 μm 4.08 μm4.25 μm 8.18 μm 90^(th) percentile, 5.61 μm 6.52 μm 6.71 μm 13.63 μm d(v, 0.9) % microsphere 2.18% 7.17% 8.23% 34.38% volumetric diameter >7μm

Results. An aggregate of microspheres will be interpreted by theparticle size analyzer as a single larger microsphere. If aggregation ofthe microspheres is being reduced, it would be reflected by a sizemeasurement that has shifted downward. Comparison of the size histogramstatistics in the table (see FIGS. 1A-1D) reveals a trend toward smallersize microspheres and thus less aggregation in the suspensions thatcontain sucrose or tert-butyl alcohol in the formulation (formulations 2& 3) over the formulation that contains neither ingredient (formulation4). Also, there appears to be an additive effect to the reduction ofmicrosphere aggregation when both sucrose and tert-butyl alcohol arepresent (formulation 1).

Microscopic inspection of formulation 1 and formulation 4 qualitativelyconfirmed the presence of a much greater degree of microsphereaggregation with formulation 4 than with formulation 1.

Example 2

The example demonstrates the ability of the amorphous sugar sucroseand/or t-butyl alcohol to reduce aggregation of solid-coredalbumin-coated polylactide microspheres during lyophilization andreconstitution.

Preparation Of Albumin-Coated Solid Polylactide Microspheres. A 6%aqueous solution was prepared from a 25% solution of USP grade humanserum albumin (HSA) by dilution with deionized water. The pH of thesolution was adjusted to 4 using 6M HCl. Separately, a 10% solution ofpoly (D,L-lactide) was prepared by dissolution of the polymer intoisopropyl acetate. The organic solution in the amount of 42 ml wasslowly incorporated with stirring into 25 ml of the prepared HSAsolution while maintaining a temperature of 30° C. The resulting coarseo/w emulsion was then circulated through a stainless steel sinteredmetal filter element. The emulsion was next diluted to 4× volume withdeionized water and then added with stirring to 400 ml of deionizedwater maintained at 30° C. Immediately upon addition of the dilutedemulsion, 1 ml of 25% glutaraldehyde and 2 ml of 1N NaOH were added tothe stirring bath. Stirring was continued for approximately 3 hoursuntil the isopropyl acetate had evaporated. After the 3 hours, 5 ml of a15% solution of poloxamer 188 was added to the microsphere suspension.The microsphere were retrieved by centrifugation and washed 3 timesusing an aqueous solution of 0.25% poloxamer 188. The size of themicrospheres were measured with a Malvern 2000 particle size analyzerand found to have a volumetric peak diameter of 4.34 microns.

Solid Polylactide Microsphere Formulation And Lyophilization. Thesuspension of solid polylactide microspheres was diluted with 0.25%poloxamer 188 to achieve a microsphere concentration of approximately2.5E+9 particles/ml. Separately, four different aqueous solutions wereprepared to serve as lyophilization excipients using ingredients and atthe concentrations (by weight) in accordance with the table below.

Lyophilization Formulation Designation Excipient Solution 1 2 3 4tert-butyl alcohol 26.25%  26.25%    0%   0% Polyethylene glycol 4.34%4.34%  4.34% 4.34%  Glycine  1.8% 1.8%  1.8% 1.8% Poloxamer 188  0.9%0.9%  0.9% 0.9% Sucrose 0.38%   0% 0.38%   0% Deionized water 66.3%66.7%  92.6% 93.0% 

The diluted microsphere suspension was next formulated with the 4prepared excipients at a ratio of 1 part suspension to 2 parts excipientsolution by weight. The resulting formulations were each dispensed into10 ml serum vials and then lyophilized to a dry cake using an FTSDura-Stop lyophilizer and capped under nitrogen.

Measurement of Particle Size. After lyophilization, vials werereconstituted in 2 ml deionized water and the size distribution of themicrospheres in the suspensions were determined using a Malvern 2000particle size analyzer. Results of the size measurements are shown inthe table below. The derived statistics in the table are based upon avolumetric frequency histogram of microsphere size and represent anaverage over three vials.

Formulation Designation 1 2 3 4 Mode Diameter 5.66 μm 5.99 μm 7.28 μm 9.48 μm Mean Diameter 6.22 μm 7.77 μm 8.06 μm 12.75 μm 90th percentile,10.57 μm  12.23 μm  13.82 μm  23.24 μm d(v, 0.9) % microsphere 12.2%17.6% 25.2% 45.4% volume >10 μm

Results. An aggregate of microspheres will be interpreted by theparticle size analyzer as a single larger microsphere. If aggregation ofthe microspheres is being reduced, it would be reflected by a sizemeasurement that has shifted downward. Comparison of the size histogramstatistics in the table (see FIGS. 2A-2D) reveals a trend toward smallersize microspheres and thus less aggregation in the suspensions thatcontain sucrose or tert-butyl alcohol in the formulation (formulations 2& 3) over the formulation that contains neither ingredient (formulation4). Also, there appears to be an additive effect to the reduction ofmicrosphere aggregation when both sucrose and tert-butyl alcohol arepresent (formulation 1).

Example 3

This example demonstrates the effect of sucrose concentration on removalof residual solvent from the core of hollow-cored microspheres.

Cyclooctane filled microspheres were prepared as described in Example 1.Separately, four aqueous solutions, with increasing sucroseconcentration, were prepared to serve as lyophilization excipients usingingredients and at concentrations (by weight) in accordance with thetable below.

Lyophilization Formulation Designation Excipient Solution 1 2 3 4tert-butyl alcohol 26.25%  26.25%  26.25%  26.25%  Polyethylene glycol4.34% 4.34% 4.34% 4.34% Glycine  1.8%  1.8%  1.8%  1.8% Poloxamer 1880.78% 0.78% 0.78% 0.78% Sucrose  0.0% 0.15% 0.38%  0.6% Deionized water66.83%  66.68%  66.45%  66.23% 

The diluted microsphere suspension was next formulated with the 4prepared excipients at a ratio of 1 part suspension to 2 parts excipientsolution by weight. The resulting formulations were each dispensed into10 ml serum vials at 3 ml/vial and then lyophilized to a dry cake usinga Virtis Ultra-35XL lyophilizer and capped under nitrogen. During thislyophilization process, the cyclooctane core of the microspheres wasremoved to render hollow nitrogen-filled microspheres.

Product vials were analyzed for residual cyclooctane by gaschromatography. The results are tabulated below.

Formulation Residual Cyclooctane Number (micrograms per vial) 1 10.6 μg2 13.4 μg 3 274 μg 4 926 μg

Example 4

This example demonstrates the effect of t-butyl alcohol concentration onremoval of residual solvent from the cores of hollow-cored microspheres.

Cyclooctane filled microspheres were prepared as described in Example 1.Separately, five aqueous solutions, with increasing tert-butyl alcoholconcentration, were prepared to serve as lyophilization excipients usingingredients and at concentrations (by weight) in accordance with thetable below.

Lyophilization Formulation Designation Excipient Solution 1 2 3 4 5tert-butyl alcohol   0%   15% 26.25%    30% 37.5% Polyethylene glycol4.34% 4.34% 4.34% 4.34% 4.34% Glycine  1.8%  1.8%  1.8%  1.8%  1.8%Poloxamer 188 0.78% 0.78% 0.78% 0.78% 0.78% Sucrose 0.38% 0.38% 0.38%0.38% 0.38% Deionized water 92.58%  77.58%  66.33%  62.58%  55.08% 

The diluted microsphere suspension was next formulated with the 5prepared excipients at a ratio of 1 part suspension to 2 parts excipientsolution by weight. The resulting formulations were each dispensed into10 ml serum vials at 3 ml/vial and then lyophilized to a dry cake usinga Virtis Ultra-35XL lyophilizer and capped under nitrogen. During thislyophilization process, the cyclooctane core of the microspheres wasremoved to render hollow nitrogen-filled microspheres.

Product vials were analyzed for residual cyclooctane by gaschromatography. The results are tabulated below.

Formulation Residual Cyclooctane Number (micrograms per vial) 1 1654 μg2 483 μg 3 152 μg 4 92 μg 5 79 μg

The foregoing description of specific embodiments and examples of theinvention have been presented for the purpose of illustration anddescription, and although the invention has been illustrated by certainof the preceding examples, it is not to be construed as being limitedthereby. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications, embodiments, and variations are possible in light of theabove teaching. It is intended that the scope of the invention encompassthe generic area as herein disclosed, and by the claims appended heretoand their equivalents.

1. An aqueous particle suspension comprising: a plurality of particlesin an amount of 0.3 to 4 mg per milliliter of suspension; one or moreamorphous sugars at a total weight to weight ratio (wt amorphous sugars:wt particles) in the range of 0.3:1 to 12:1 for hollow-cored particles(based upon the shell weight of the particles) or 0.02:1 to 0.86:1 forsolid-cored particles; and t-butyl alcohol at a weight to weight ratio(t-butyl alcohol:particles) in the range of 30:1 to 600:1 forhollow-cored particles or 2:1 to 43:1 for solid-cored particles.
 2. Theparticle suspension of claim 1, wherein said amorphous sugar is selectedfrom the group consisting of sucrose, trehalose and lactose andcombinations thereof.
 3. The particle suspension of claim 1, furthercomprising a surfactant at a weight to weight ratio(surfactant:particles) in the range of 0.01:1 to 60:1 for hollow-coredparticles or 0.01:1 to 4.5:1 for solid cored particles.
 4. The particlesuspension of claim 1, further comprising a water soluble polymer at aweight to weight ratio (water soluble polymer:particles) in the range of6:1 to 300:1 for hollow-cored particles or 0.4:1 to 22:1 for solid-coredparticles.
 5. The particle suspension of claim 1, further comprising anosmolality adjusting agent at a weight to weight ratio (agent:particles)in the range of 0.01:1 to 75:1 for hollow-cored particles or 0.01:1 to6:1 for solid-cored particles.
 6. The particle suspension of claim 1, inwhich the particles are hollow-cored, and the composition furthercomprises: a water soluble polymer at a weight to weight ratio (polymer:particle) in the range of 6:1 to 300:1; a surfactant at a weight toweight ratio (surfactant:particles) in the range of 0:1 to 60:1; and anosmolality adjusting agent at a weight to weight ratio (agent:particles)in the range of 0:1 to 75:1.
 7. The particle suspension of claim 4 or 6,wherein said water soluble polymer is selected from the group consistingof medium molecular weight polyethylene glycols, low to medium molecularweight polyvinylpyrrolidones, and combinations thereof.
 8. The particlesuspension of claim 3 or 6, wherein said surfactant is a poloxamer. 9.The particle suspension of claim 5 or 6, wherein said osmolalityadjusting agent is glycine.
 10. The particle suspension of claim 1 or 6,wherein said particles are hollow-cored bilayered particles.
 11. Theparticle suspension of claim 10, wherein said hollow-cored bilayeredparticles comprise a shell enclosing a hollow core, wherein the shellcomprises an inner layer of a biodegradable polymer and an outer layerof a cross-linked amphiphilic material.
 12. The particle suspension ofclaim 11, wherein said outer layer comprises glutaraldehyde crosslinkedalbumin.
 13. The particle suspension of claim 11, wherein said innerlayer comprises poly(D,L-lactide).
 14. The particle suspension of claim11, wherein said hollow-cored bilayered particles have an averagediameter in the range of approximately 1 to 10 micrometers.
 15. Theparticle suspension of claim 11, wherein said hollow-cored bilayeredparticles have an average diameter in the range of approximately 200 to800 nanometers.
 16. The particle suspension of claim 1, wherein: saidinner layer comprises poly(,L-lactide); said outer layer comprisesglutaraldehyde-linked albumin; and said hollow cored bilayered particleshave an average diameter in the range of approximately 1 to 10micrometers.
 17. The particle suspension of claim 1, wherein: said innerlayer comprises poly(D,L-lactide); said outer layer comprisesglutaraldehyde-linked albumin; and said hollow-cored bilayered particleshave an average diameter in the range of approximately 200 to 800nanometers.
 18. A particle composition prepared by lyophilizing todryness a particle suspension according to any one of claims 1-17.
 19. Adry composition comprising: a plurality of hollow-cored microparticlesin an amount of 3.3 wt %; polyethylene glycol 3350 in an amount of 56.6wt %; poloxamer 188 in an amount of 11.7 wt %; sucrose in an amount of4.9 wt %; and glycine in an amount of 23.5%.
 20. The dry composition ofclaim 19 in which the hollow-cored microparticles comprise a bilayeredshell, the bilayered shell comprising an inner layer of poly(D,L)lactideand an outer layer of glutaraldehyde crosslinked albumin.
 21. The drycomposition of claim 19 or 20 in which the hollow core of thehollow-cored microparticles is filled with a gas or mixture of gases.22. The dry composition of claim 21 in which the gas is nitrogen. 23.The dry composition of claim 22 which is packaged in a stoppered glassvial that is sealed under nitrogen.
 24. The dry composition of claim 23in which the glass vial contains 2.5 to 7 mg microparticles, 86.7 mgpolyethylene glycol 3350, 18 mg poloxamer 188, 7.5 mg sucrose and 36 mgglycine.
 25. A method of making a dry composition of polymeric and/orproteinaceous particles comprising lyophilizing to dryness a particlesuspension according to any one of claims 1-17.
 26. A method of making adry composition of hollow-cored bilayered microspheres suitable for usein ultrasound imaging, comprising the steps of: (i) emulsifying anorganic solution comprising 0.9 wt % poly(D,L-lactide), 12.9 wt %cyclooctane and 86.2 wt % isopropyl acetate with a first aqueoussolution comprising 5 wt % human serum albumin to yield an oil-in-wateremulsion; (ii) diluting the emulsion 3 to 18-fold into a second aqueoussolution comprising glutaraldehyde in an amount sufficient to yield aweight to weight ratio (wt glutaraldehyde:wt albumin) of 0.2:1; (iii)stirring the result of step (ii) until the isopropyl acetate issubstantially removed; (iv) adding poloxamer 188 to the result of (iii)in a amount sufficient to yield a concentration of 0.25 wt %; (v)diafiltering the result of (iv) against an aqueous solution of poloxamer188 (0.25 wt %) to remove glutaraldehyde and unassociated albumin; (vi)diluting the result of (v) with an aqueous solution of poloxamer 188(0.25 wt %) to yield a suspension having a microsphere concentration of2.5 to 7 mg microspheres (based on their shell weight) per gram ofsuspension; (vii) adding to the result of step (vi) an aqueous excipientsolution comprising 26.25 wt % t-butyl alcohol, 0.375 wt % sucrose, 1.8wt % glycine, 4.335 wt % polyethylene glycol 3350 and 0.775 wt %poloxamer 188 in an amount sufficient to yield an aqueous suspension ofmicroparticles comprising 1.7 mg of microparticles (based upon themicrosphere shell weight) per gram of suspension; and (viii)lyophilizing to dryness the aqueous microparticle suspension of step(vii).
 27. The method of claim 26, further comprising the step ofback-filling the microspheres with nitrogen gas.